SPONSORED RESEARCH AND DEVELOPMENT [Not Applicable]
This invention pertains to a biosensor for detecting and/or quantifying analytes. More particularly, this invention pertains to a biosensor based on a detection element that is a single macromolecule spanning two electrodes.
Biosensors are devices that can detect and/or quantify analytes using known interactions between a targeted analyte and a binding agent that is typically a biological macromolecule such as an enzyme, receptor, nucleic acid, protein, lectin, or antibody. Biosensors have applications in virtually all areas of human endeavor. For example, biosensors have utility in fields as diverse as blood glucose monitoring for diabetics, the recognition of poisonous gas and/or explosives, the detection of chemicals commonly associated with spoiled or contaminated food, genetic screening, environmental testing, and the like.
Biosensors are commonly categorized according to two features, namely, the type of macromolecule utilized in the device and the means for detecting the contact between the binding agent and the targeted analyte. Major classes of biosensors include enzyme (or catalytic) biosensors, immunosensors and DNA biosensors.
Enzyme (or catalytic) biosensors typically utilize one or more enzymes as the macromolecule and take advantage of the complimentary shape of the selected enzyme and the targeted analyte. Enzymes are proteins that perform most of the catalytic work in biological systems and are known for highly specific catalysis. The shape and reactivity of a given enzyme limits its catalytic activity to a very small number of possible substrates. Enzyme biosensors rely on the specific chemical changes related to the enzyme/analyte interaction as the means for recognizing contact with the targeted analyte. For example, upon interaction with an analyte, an enzyme biosensor may generate electrons, a colored chromophore or a change in pH as the result of the relevant enzymatic reaction. Alternatively, upon interaction, with an analyte, an enzyme biosensor may cause a change in a fluorescent or chemiluminesceint signal that can be recorded by an appropriate detection system.
Immunosensors utilize antibodies as binding agents. Antibodies are protein molecules that generally do not perform catalytic reactions, but specifically bind to particular xe2x80x9ctargetxe2x80x9d molecules (antigens). Antibodies are quite specific in their interactions and, unlike most enzymes, they are capable of recognizing and selectively binding to very large bodies such as single cells. Thus, in addition to detection of small analytes, antibody-based biosensors allow for the identification of certain pathogens such as dangerous bacterial strains.
DNA biosensors typically utilize the complimentary nature of the DNA or RNA double-strands and are designed for the specific detection of particular nucleic acids. A DNA biosensor sensor generally uses a single-stranded DNA as the binding agent. The nucleic acid material in a given test sample is placed into contact with the binding agent under conditions where the biosensor DNA and the target nucleic acid analyte can form a hybrid duplex. If a nucleic acid in the test sample is complementary to a nucleic acid used in the biosensor, the two interact/bind. The interaction can be monitored by various means such as a change in mass at the sensor surface or the presence of a fluorescent or radioactive signal. In alternative arrangements, the target nucleic acid(s) are bound to the sensor and contacted with labeled probes to allow for identification of the sequence(s) of interest.
While the potential utility for biosensors is great and while hundreds of biosensors have been described in patents and in the literature, actual commercial use of biosensors remains limited. Aspects of biosensors that have limited their commercial acceptance include a lack the sensitivity and/or speed of detection necessary to accomplish certain tasks, problems with long term stability, difficulty miniaturizing the sensor, and the like. In addition, a number of biosensors must be pre-treated with salts and/or enzyme cofactors, a practice that is inefficient and bothersome.
This invention pertains to the development of a novel molecular sensing apparatus (biosensor) and to methods of use thereof. In preferred embodiments, the sensing apparatus comprises a first electrode, a second electrode, an insulator between the first electrode and the second electrode; and a binding agent (e.g. a biological macromolecule) connecting the first electrode and the second electrode. In particularly preferred embodiments, the binding agent is attached to the electrode in a manner that permits charge to flow from the electrode to the binding agent or from the binding agent to the electrode. Preferred binding agents include, but are not limited to, biological macromolecules (e.g. a nucleic acid, a protein, a polysaccharide, a lectin, a lipid, etc.) with a nucleic acid being most preferred. While the nucleic acid can be essentially any length, preferred nucleic acids range in length from about 5 nucleotides to about 5,000 nucleotides, more preferably from about 8 nucleotides to about 1,000 nucleotides or 500 nucleotides, still more preferably from about 10 nucleotides to about 300 nucleotides, and most preferably from about 15, 20, 25, 30 or 50 nucleotides to about 100 nucleotides or 150 nucleotides in length. Typically, the nucleic acid is of sufficient length to specifically hybridize to a target nucleic acid in a complex population of nucleic acids (e.g. total genomic DNA) under stringent conditions.
In preferred embodiments, the biological macromolecule is functionalized with a chemical group thereby facilitating the attachment of the macromolecule to the electrode(s). Preferred chemical groups include, but are not limited to a sulfate, a sulfhydryl, an amine, an aldehyde, a carboxylic acid, a phosphate, a phosphonate, an alkene, an alkyne, a hydroxyl group, a bromine, an iodine, a chlorine, a light-activatable (labile) group, a group activatable by an electric potential, and the like. In certain embodiments, the biological macromolecule is functionalized with a second biological macromolecule (e.g. a receptor, a receptor ligand, an antibody, an epitope, a nucleic acid, a lectin, a sugar, and the like). In preferred embodiments, however, such second biological macromolecules exclude nucleic acids.
Preferred insulators are insulators having a resistivity greater than about 10xe2x88x923 ohm-meters, more preferably greater than about 10xe2x88x922 ohm-meters, and most preferably greater than about 10xe2x88x921, 1, or 10 ohm-meters. Suitable insulators include, but are not limited to SiO2, TiO2, ZrO2, quartz, porcelain, ceramic, polystyrene, Teflon (other high-resistivity plastics), an insulating oxide or sulfide of a transition metal in the periodic table of the elements, and the like.
In certain preferred embodiments, the first electrode and the second electrode are separated by a distance in the range of 1 to 1010 Angstroms. Typically the first electrode and the second electrode are separated by a distance less than about 300 Angstroms, preferably less than about 150 Angstroms, more preferably less than about 70 Angstroms, and most preferably less than about 50 angstroms.
In certain embodiments, the first electrode and/or the second electrode have a resistivity of less than about 10xe2x88x922 ohm-meters, preferably less than about 10xe2x88x923 ohm-meters, more preferably less than about 10xe2x88x924 ohm-meters, and most preferably less than about 10xe2x88x925, or 10xe2x88x926 ohm-meters. Particularly preferred electrodes comprise a material such as ruthenium, osmium, cobalt, rhodium, rubidium, lithium, sodium, potassium, vanadium, cesium, beryllium, magnesium, calcium, chromium, molybdenum, silicon, germanium, aluminum, iridium, nickel, palladium, platinum, iron, copper, titanium, tungsten, silver, gold, zinc, cadmium, indium tin oxide, carbon, or a carbon nanotube. In certain preferred embodiments, the first electrode is functionalized to contain a chemical group that can be derivatized or crosslinked (e.g., a sulfate, a sulfhydryl, an amine, an aldehyde, a carboxylic acid, a phosphate, a phosphonate, an alkene, an alkyne, a hydroxyl group, a bromine, an iodine, a chlorine, a light-activatable group, a group activatable by an electric potential, etc.). The first and/or second electrode can bear a self-assembled monolayer (SAM). Particularly preferred SAMs comprise a compound selected from the group consisting of an alkanethiol, a phospholipid, a bola amphiphile, and an oligo(phenylenevinylene).
In a particularly preferred embodiment, the biological macromolecule is attached to the first and/or to the second electrode directly by a thiol group or through a linker bearing a thiol group. In another particularly preferred embodiment, the biological macromolecule is attached to the first and/or to the second electrode directly by a phosphonate or through a linker bearing a phosphonate. In preferred embodiments, the biological macromolecule is attached to the first and/or to the second electrode by a linker (e.g., DFDNB, DST, ABH, ANB-NOS, EDC, NHS-ASA, SIA, oligo(phenylenevinylene), etc.).
The apparatus can further comprise a substrate (other than the electrode and/or insulator) where the first electrode and the second electrode are integrated with the substrate. In certain embodiments, the first electrode and the second electrode are integrated with the insulator to form a substrate. The electrodes can be formed in essentially any desired shape (e.g. convex, concave, textured, corrugated, patterned uniformly, randomly patterned, etc.). Certain preferred electrode orientations include annular, planar, and orthogonal. In certain embodiments, the first electrode comprises a first surface and a second electrode comprises a second surface where the first surface and the second surface are not co-planar.
The apparatus can comprise a plurality of electrode pairs. Thus, in certain embodiments, the first electrode and the second electrode comprise a first electrode pair, and the molecular sensing apparatus further comprises a second electrode pair comprising a second first electrode and a second second electrode. In certain embodiments, the apparatus comprises at least 3, preferably at least 10 or 20, more preferably at least 50, 100, or 1,000, and most preferably at least 10,000 or at least 1,000,000 electrode pairs.
In certain embodiments, the apparatus further comprises a measurement device electrically coupled to the first electrode and to the second electrode of at least one said electrode pair. Preferred measurement devices measure an electromagnetic property selected from the group consisting direct electric current, alternating electric current, permitivity, resistivity, electron transfer, electron tunneling, electron hopping, electron transport, electron conductance, voltage, electrical impedance, signal loss, dissipation factor, resistance, capacitance, inductance, magnetic field, electrical potential, charge and magnetic potential. One particularly preferred measurement device is a potentiostat.
The apparatus can further comprise an electrical circuit electrically coupled to the first electrode and the second electrode. One such circuit comprises an electrical signal gating system (e.g. a CMOS gating system), and/or a voltage source, and/or a multiplexor, and/or a computer.
In certain embodiments, the electrodes comprising the first and second electrode pairs have attached the same (species of) biological macromolecule. In certain embodiments, different electrode pairs, have attached different biological molecules.
In certain embodiments, the first electrode and/or the second electrode comprise a semi-conducting material. Preferred semiconducting materials have a resistivity ranging from about 10xe2x88x926 ohm-meters to about 10xe2x88x927 ohm-meters. Preferred semiconducting materials include, but are not limited to silicon, dense silicon carbide, boron carbide, Fe3O4, germanium, silicon germanium, silicon carbide, tungsten carbide, titanium carbide, indium phosphide, gallium nitride, gallium phosphide, aluminum phosphide, aluminum arsenide, mercury cadmium telluride, tellurium, selenium, ZnS, ZnO, ZnSe, CdS, ZnTe, GaSe, CdSe, CdTe, GaAs, InP, GaSb, InAs, Te, PbS, InSb, PbTe, PbSe, and tungsten disulfide.
In one embodiment, the apparatus comprises: a first electrode having a first surface; a second electrode having a second surface coplanar to the first surface; an insulator between said first surface and said second surface; and a nucleic acid joining the first electrode to said second electrode.
This invention also provides a method of making a molecular sensing apparatus. In certain embodiments, the method comprises: providing a first electrode and a second electrode separated by an insulator; contacting the first and the second electrode with a first solution comprising a biological macromolecule (e.g., a nucleic acid); placing a charge on the first electrode to attract the biological macromolecule to the first electrode where the macromolecule attaches to the first electrode to form an attached macromolecule; and placing a charge on the second electrode to attract a portion of the attached macromolecule to the second electrode to attach the macromolecule to the second electrode. Preferred macromolecules, electrodes, electrode configurations, insulators, measurement devices, circuits, and the like, include, but are not limited to those described above. Where the apparatus comprises multiple electrode pairs, the method can further comprise contacting a second electrode pair with a second solution comprising a second biological macromolecule; placing a charge on the first electrode of the second electrode pair to attract the second biological macromolecule to the first electrode of the second electrode pair whereby the second biological macromolecule attaches to said first electrode to form an attached second macromolecule; and placing a charge on the second electrode of said second electrode pair to attract a portion of said attached second macromolecule to attach said second macromolecule to said second electrode of said second electrode pair. The first and second solution can be the same or different. Similarly, the first biological macromolecule and the second biological macromolecule can be the same or different.
In still another embodiment, this invention provides a method of detecting an analyte. The method involves i) providing a molecular sensing apparatus comprising a first electrode and a second electrode separated by an insulator where said first electrode has a biological macromolecule attached thereto; ii) contacting the attached macromolecule with said analyte whereby said analyte binds to said macromolecule thereby forming a macromolecule/analyte complex; iii) placing a charge on said second electrode to attract a portion of said bound analyte to said second electrode where said second analyte is bound to the second electrode such that the macromolecule/analyte complex forms a connection between the first electrode and the second electrode; and iv) detecting the connection between said first and said second electrode. In certain embodiments, the providing comprises: contacting the first electrode with a first solution comprising the biological macromolecule; and placing a charge on the first electrode whereby the charge attracts the biological macromolecule to the electrode and the biological macromolecule attaches to the electrode. Where multiple electrode pairs are present, the method can involve repeating these steps for each electrode pair. The xe2x80x9cplacing a chargexe2x80x9d can, optionally involve placing a charge on the first electrode opposite to the charge on the second electrode. In certain embodiments, the xe2x80x9cdetectingxe2x80x9d comprises detecting an electromagnetic property selected from the group consisting of direct electric current, alternating electric current, permittivity, resistivity, electron transfer, electron tunneling, electron hopping, electron transport, electron conductance, voltage, electrical impedance, signal loss, dissipation factor, resistance, capacitance, inductance, magnetic field, electrical potential, charge, and magnetic potential. Preferred macromolecules, electrodes, electrode configurations, insulators, measurement devices, circuits, and the like, include, but are not limited to those described above.
In still another embodiment, this invention provides a method of detecting an analyte, where the method involves: i) providing a molecular sensing apparatus comprising a first electrode and a second electrode separated by an insulator where the first electrode has a first biological macromolecule attached thereto and the second electrode has a second biological macromolecule attached thereto; ii) contacting the first attached macromolecule and the second attached macromolecule with the analyte whereby said analyte binds to the first macromolecule and to the second macromolecule thereby forming a macromolecule/analyte complex forming a connection between said first electrode and said second electrode; and iii) detecting the connection between said first and said second electrode. In certain embodiments, the xe2x80x9cprovidingxe2x80x9d comprises contacting the first electrode with a first solution comprising the first biological macromolecule; and placing a charge on the first electrode whereby the charge attracts the first biological macromolecule to the electrode and the biological macromolecule attaches to the electrode. Similarly, in certain embodiments, the xe2x80x9cprovidingxe2x80x9d comprises contacting the second electrode with a solution comprising the second biological macromolecule; and placing a charge on the second electrode whereby the charge attracts the second biological macromolecule to the second electrode and the second biological macromolecule attaches to the second electrode. In certain embodiments, the xe2x80x9cdetectingxe2x80x9d comprises detecting an electromagnetic property selected from the group consisting of direct electric current, alternating electric current, permitivity, resistivity, electron transfer, electron tunneling, electron hopping, electron transport, electron conductance, voltage, electrical impedance, signal loss, dissipation factor, resistance, capacitance, inductance, magnetic field, electrical potential, charge, and magnetic potential. Preferred macromolecules, electrodes, electrode configurations, insulators, measurement devices, circuits, and the like, include, but are not limited to those described above.
This invention provides still another method of detecting an analyte. The method involves i) providing a molecular sensing apparatus comprising a first electrode and a second electrode separated by an insulator where a biological macromolecule forms a connection between the first electrode and the second electrode; ii) detecting the connection between the first and the second electrode; iii) contacting the biological macromolecule (binding agent) with the analyte whereby the analyte binds to the macromolecule thereby forming a macromolecule/analyte complex; and iv) detecting a difference in the connection between the first electrode and the second electrode. In certain embodiments, the xe2x80x9ccontactingxe2x80x9d comprises placing a charge on the first and/or the second electrode whereby the charge attracts the analyte to the biological macromolecule. In certain embodiments, the xe2x80x9cprovidingxe2x80x9d comprises contacting the first electrode with a first solution comprising the biological macromolecule; and placing a charge on the first electrode whereby the charge attracts the biological macromolecule to the electrode and the biological macromolecule attaches to the electrode; and placing a charge on the second electrode to attract a portion of the bound macromolecule to the second electrode where the macromolecule is bound to the second electrode such that the macromolecule forms a connection between the first electrode and said second electrode. In certain embodiments, the xe2x80x9cplacing a chargexe2x80x9d comprises placing a charge on the first electrode opposite to the charge on the second electrode. The xe2x80x9cdetectingxe2x80x9d can comprise detecting an electromagnetic property selected from the group consisting of direct electric current, alternating electric current, permitivity, resistivity, electron transfer, electron tunneling, electron hopping, electron transport, electron conductance, voltage, electrical impedance, signal loss, dissipation factor, resistance, capacitance, inductance, magnetic field, electrical potential, charge and magnetic potential. In particularly preferred embodiments, the biological macromolecule is attached to the first electrode by an electrically conductive linker. In certain embodiments, the binding agent is a nucleic acid and the analyte is a protein or a protein complex. Preferred macromolecules, electrodes, electrode configurations, insulators, measurement devices, circuits, and the like, include, but are not limited to those described above.
Any of the methods and devices described herein include embodiments where the binding agents are not joined to the first electrode and/or the second electrodes a second or third nucleic acid. Thus, in such embodiments, where the binding agent is a nucleic acid, a single nucleic acid molecule spans the first and second electrode and linkers or functional groups, if present, are not themselves nucleic acids.
Definitions
The term xe2x80x9cbiosensorxe2x80x9d refers to a sensor that uses a biological macromolecule (e.g. nucleic acid, carbohydrate, protein, antibody, etc.) to specifically recognize/bind to a target analyte. The term xe2x80x9cmolecular sensing apparatusxe2x80x9d is used interchangeably with the term xe2x80x9cbiosensorxe2x80x9d.
The term xe2x80x9cbiological macromoleculexe2x80x9d as used herein refers to a biological molecule such as a nucleic acid, protein, antibody, carbohydrate, polysaccharide, lipid, and the like.
The term xe2x80x9celectrically conductivexe2x80x9d wherein used with reference to a linker, molecule or molecular complex refers to the ability of that linker, molecule or molecular complex to pass charge through itself. Preferred electrically conductive molecules have a resistivity lower than about 10xe2x88x923 more preferably lower than about 10xe2x88x924, and most preferably lower than about 10xe2x88x926 or 10xe2x88x927 ohm-meters.
The term xe2x80x9celectrically coupledxe2x80x9d binding agent and an electrode refers to an association between that binding agent and the electrode such that electrons can move from the binding agent to the electrode or from the electrode to the binding agent. Electrical coupling can include direct covalent linkage between the binding agent and the electrode, indirect covalent coupling (e.g. via a linker), direct or indirect ionic bonding between the binding agent and the electrode, or other bonding (e.g. hydrophobic bonding). In addition, no actual bonding may be required and the binding agent can simply be contacted with the electrode surface.
The term xe2x80x9csensor elementxe2x80x9d as used herein refers to a pair of electrodes (e.g. first electrode 10 and second electrode 12) and associated binding agent(s) 14 that, when bound by an analyte form a molecular complex that spans the pair of electrodes.
The terms xe2x80x9cpolypeptidexe2x80x9d, xe2x80x9cpeptidexe2x80x9d and xe2x80x9cproteinxe2x80x9d are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
The term xe2x80x9cnucleic acidxe2x80x9d as used herein refers to a deoxyribonucleotide or ribonucleotide in either single- or double-stranded form. The term encompasses nucleic acids, i.e., oligonucleotides, containing known analogues of natural nucleotides which have similar or improved binding properties, for the purposes desired, as the reference nucleic acid. The term also encompasses nucleic-acid-like structures with synthetic backbones. DNA backbone analogues provided by the invention include phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3xe2x80x2-thioacetal, methylene(methylimino), 3xe2x80x2-N-carbamate, morpholino carbamate, and peptide nucleic acids (PNAs); see Oligonucleotides and Analogues, a Practical Approach, edited by F. Eckstein, IRL Press at Oxford University Press (1991); Antisense Strategies, Annals of the New York Academy of Sciences, Volume 600, Eds. Baserga and Denhardt (NYAS 1992); Milligan (1993) J. Med. Chem. 36:1923-1937; Antisense Research and Applications (1993, CRC Press). PNAs contain non-ionic backbones, such as N-(2-aminoethyl) glycine units. Phosphorothioate linkages are described in WO 97/03211; WO 96/39154; Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197. Other synthetic backbones encompasses by the term include methyl-phosphonate linkages or alternating methylphosphonate and phosphodiester linkages (Strauss-Soukup (1997) Biochemistry 36: 8692-8698), and benzylphosphonate linkages (Samstag (1996) Antisense Nucleic Acid Drug Dev 6: 153-156). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide primer, probe and amplification product.
The term xe2x80x9cantibodyxe2x80x9d refers to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof which specifically bind and recognize an analyte (antigen). The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one xe2x80x9clightxe2x80x9d (about 25kD) and one xe2x80x9cheavyxe2x80x9d chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.
Antibodies exist e.g., as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)xe2x80x22, a dimer of Fab which itself is a light chain joined to VHxe2x80x94CH1 by a disulfide bond. The F(ab)xe2x80x22 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)xe2x80x22 dimer into an Fabxe2x80x2 monomer. The Fabxe2x80x2 monomer is essentially an Fab. with part of the hinge region (see, Fundamental Immunology, Third Edition, W. E. Paul, ed., Raven Press, N.Y. 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments maybe synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv), and those found in display libraries (e.g. phage display libraries).
The phrases xe2x80x9chybridizing specifically toxe2x80x9d or xe2x80x9cspecific hybridizationxe2x80x9d or xe2x80x9cselectively hybridize toxe2x80x9d, refer to the binding, duplexing, or hybridizing of a nucleic acid molecule preferentially to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
The term xe2x80x9cstringent conditionsxe2x80x9d refers to conditions under which a probe will hybridize preferentially to its target sequence, and to a lesser extent to, or not at all to, other sequences. xe2x80x9cStringent hybridizationxe2x80x9d and xe2x80x9cstringent hybridization wash conditionsxe2x80x9d in the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations are sequence dependent, and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biologyxe2x80x94Hybridization with Nucleic Acid Probes part I chapter 2 Overview of principles of hybridization and the strategy of nucleic acid probe assays, Elsevier, N.Y. Generally, highly stringent hybridization and wash conditions are selected to be about 5xc2x0 C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the Tm for a particular probe.
An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formamide with 1 mg of heparin at 42xc2x0 C., with the hybridization being carried out overnight. An example of highly stringent wash conditions is 0.15 M NaCl at 72xc2x0 C. for about 15 minutes. An example of stringent wash conditions is a 0.2xc3x97SSC wash at 65xc2x0 C. for 15 minutes (see, Sambrook et al. (1989) Molecular Cloningxe2x80x94A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, N.Y., (Sambrook et al.) supra for a description of SSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example of a medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is 1xc3x97SSC at 45xc2x0 C. for 15 minutes. An example of a low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4-6xc3x97SSC at 40xc2x0 C. for 15 minutes. In general, a signal to noise ratio of 2xc3x97 (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization. Nucleic acids which do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
In one particularly preferred embodiment, stringent conditions are characterized by hybridization in 1 M NaCl, 10 mM Tris-HCl, pH 8.0, 0.01% Triton X-100, 0.1 mg/ml fragmented herring sperm DNA with hybridization at 45xc2x0 C. with rotation at 50 RPM followed by washing first in 0.9 M NaCl, 0.06 M NaH2PO4, 0.006 M EDTA, 0.01% Tween-20 at 45xc2x0 C. for 1 hr, followed by 0.075 M NaCl, 0.005 M NaH2PO4, 0.5 mM EDTA at 45xc2x0 C. for 15 minutes.
A xe2x80x9chigh resistivity plasticxe2x80x9d refers to a plastic with a resistivity greater than about 10xe2x88x923 ohm-meters, more preferably greater than about 10xe2x88x922 ohm-meters, and most preferably greater than about 10xe2x88x921, 1 or 10 ohm-meters.